Amyloid fibril formation has been implicated in approximately 25 different diseases including Type 2 diabetes, Alzheimer's disease and Parkinson's disease. The proteins involved in each of these diseases show no sequence or structural homology. The proteins like Amylin associated with Type 2 diabetes and Amyloid Beta (Aβ) associated with Alzheimer's disease are natively unstructured. The aggregation of these proteins initially involves a structural rearrangement of monomers to form the nucleus, which then serves as a template for the addition of monomers/oligomers for fibril growth. The rates of nucleus formation as well as the growth of fibrils vary from peptide to peptide and depend on the conditions used.
The manifestation of prefibrillar and fibrillar species comprising amyloid proteins has been shown to be toxic to cells. In addition to cell death, aggregation of these normally soluble proteins makes them unavailable for the execution of their normal physiological functions. The association of these aggregates with pathogenesis of the disease has stimulated great interest in searching for molecules that can inhibit their formation. Such treatments available can either slow down the progression of the disease or simply manage the symptoms. However an effective therapeutic aid that can cure Alzheimer's disease or amyloid formation in Type 2 diabetes still remains to be established. During the past decade, several small molecules have been discovered that decrease the amyloid load in the brain and seen to partially reverse the cognitive impairment. They function either by preventing the association of the protein monomers or interfere with the pathways involved in generating the amyloidogenic peptides like Aβ. Several chaperones such as heat shock proteins (Hsps) and small heat shock proteins (sHsps) have also been shown to inhibit aggregation. Hsp 104 inhibits the fibrillization of Sup35 prion conformers. Several members of sHsps like αB-crystallin, Hsp27, Hsp20 and HspB8 bind to Aβ40 and Aβ42 and completely inhibit the aggregation of Aβ40 into mature fibrils. The small heat shock proteins bind to prefibrillar species and inhibit their progression to amyloid fibrils (Wilhelmus et al., Brain Research, 1089, 67-78, 2006; Wilhelmus et al., Acta Neuropathol., 111, 139-149, 2006).
Calnuc or Nucleobindin 1 (NUCB1) is a 55 kDa protein, which was first reported to be a growth and differentiation factor associated with lupus syndrome (Kanai et al., Immunol. Lett., 32, 43-48, 1992). Calnuc acquires its name from its DNA-binding and calcium binding ability. Its domain structure comprises, from N-terminus to C-terminus, a signal sequence at its N-terminus followed by a DNA binding domain of basic residues, an N-terminal proximal EF hand domain comprising a helix-loop-helix motif, an intervening acidic region, a second C-terminal proximal EF hand domain comprising a helix-loop-helix motif, and a leucine zipper domain (Miura et al., Biochem. Biophys. Res. Commun., 187, 375-380, 1992). Both the DNA binding domain and leucine zipper are crucial for binding of NUCB1 to DNA.
NUCB1 has been postulated to be involved in several important cellular functions. However, the pathways associated with the functionality of NUCB1, which require its conservation across the animal kingdom, still remain to be deciphered. Since its discovery, NUCB1 has been reported to be widely expressed in cells and tissues and is conserved from flies to humans (Kawano et al., Eur. J. Cell Biol. 79, 208-217, 2000). This suggests that NUCB1 is an essential calcium binding protein present in the eukaryotic genome. NUCB1 is primarily a golgi resident protein found in both cytosolic and membrane fractions (Lin et al., J. Cell Biol., 141, 1515-1527, 1998). 45Ca2+-binding assays with golgi fractions revealed NUCB1 to be the major calcium binding protein in golgi. In fact, NUCB1 shows high homology with the ER resident protein Calreticulin and displays similar properties suggesting it has a role in both calcium storage and homeostasis in golgi. Osteocytes and osteoblasts have also been reported to produce NUCB1 where it is thought to function as a modulator of matrix maturation during mineralization process in the bone (Petersson et al., Bone, 34, 949-60, 2004). The calcium binding ability of NUCB1 was found to cause accumulation and transport of Ca2+ ions to the mineralization front before hydroxyapatite deposition in bones (Somogyi et al., Calcif Tissue Int., 74, 3 66-76, 2004).
The structural basis of NUCB1 calcium binding activity has also been studied. Miura et al., reported that a C-terminal deletion of 166 residues of NUCB1 that contained only the N-terminal EF hand domain could bind calcium while a C-terminal deletion of 208 residues of NUCB1 that lacked both EF hand domains could not bind calcium (Miura et al., Biochem Biophys Res Commun. 1994 Mar. 30; 199(3):1388-93). Lin et al., disclosed an N-terminally truncated NUCB1 mutant where both EF hand loop domains and the intervening acidic region had been deleted and speculated that other acidic regions of the C-terminal domain of NUCB1 may contain low affinity calcium binding sites (Lin et al., J. Cell Biol. 141 (7): 1515. (1998). Lin et al. also subsequently reported that deletion of the entire N-terminal EF-hand (EF-1) loop domain or deletion of both EF loop hand domains and intervening acidic region of a Rat NUCB1-GFP fusion protein eliminated calcium binding (Lin et al., J Cell Biol. 1999 Apr. 19; 145(2):279-89). This same report by Lin et al. further suggested that the Rat NUCB1 protein had a single high affinity, low capacity calcium binding domain corresponding to the N-terminal (EF-1) domain.
As a first step in understanding the ubiquitous nature of NUCB1, the pathway of secretion for this protein was investigated through pulse-chase experiments. It was found that NUCB1 is synthesized in the ER and then transported to the Golgi where it resides for more than 12 hours. In the Golgi, NUCB1 undergoes O-glycosylation and sulfation and is then secreted into the extracellular medium via the constitutive-like pathway (Lavoie et al., Mol. Endocrinol., 16, 2462-74, 2002). Interactions with Cox-1 and Cox-2 isozymes have been shown to be involved in the retention of NUCB1 in the ER (Ballif et al., Proc. Natl. Acad. Sci., 93, 5544-9, 1996). NUCB1 has also been shown to interact with the Gαi; and Gαs subunits of heterotrimeric G-proteins and has been postulated to participate in regulating downstream signaling (Lavoie et al., Mol. Endocrinol., 16, 2462-74 2002).
Recent studies have suggested that overexpression of NUCB1 down-regulates the mRNA production of Amyloid precursor protein (APP) and inhibits its biosynthesis (Lin et al., J. Neurochem., 100, 1505-14, 2007). Aggregation of Aβ isoforms generated from the sequential proteolytic cleavage of APP by β-(Beta Amyloid Cleaving Enzyme-1) and γ-secretase has been well characterized in Alzheimer's disease. In addition, abnormal calcium homeostasis has also been observed in the brains of demented patients. One study indicated that: i) NUCB1 binds to APP in a calcium dependent manner where binding is inhibited by Ca+2; ii) NUCB1 co-localizes with APP in vivo; iii) NUCB1 regulates APP protein levels by affecting APP synthesis; and that iv) the expression level of NUCB1 is decreased in the brains of Alzheimer's disease patients by 50% (Lin et al., J. Neurochem., 100, 1505-14, 2007). Nonetheless, Lin et al., (J. Neurochem., 100, 1505-14, 2007) did not indicate that NUCB1 could disaggregate amyloid fibrils or inhibit amyloid fibril formation.